The ability of cells to adhere to one another plays a critical role in development, normal physiology, and disease processes. This ability is mediated by adhesion molecules, generally glycoproteins, expressed on cell membranes. Often, an adhesion molecule on one cell type will bind to another adhesion molecule expressed on a different cell type, forming a receptor counter-receptor pair. Three very important classes of adhesion molecules are the integrins, selecting, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425 (1990); Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992), all of which are incorporated herein by reference in their entirety for all purposes). These molecules are especially vital to the interaction of leukocytes and platelets with themselves and with the extracellular matrix and vascular endothelium.
Integrins are heterodimeric transmembrane glycoproteins consisting of an .alpha. chain (120-180 kD) and a .beta. chain (90-110 kD), generally having short cytoplasmic domains. The .alpha. subunits all share sequence homology and motifs with each other, as do the .beta. subunits. The three known integrins containing the .beta. subunit designated .beta..sub.2 are important to the function of T cells, neutrophils and monocytes. LFA-1 (.alpha..sub.L.beta..sub.2) is widely distributed on lymphocytes, granulocytes and monocytes. Its counter-receptor is ICAM-1 (and perhaps of lesser importance, ICAM-2) an Ig family molecule which is expressed on many cells including leukocytes and is up-regulated on vascular endothelium by cytokines such as TNF and IL-1. Blocking LFA-1 on T cells with antibodies to either the .alpha. or .beta. subunit strongly inhibits adhesion-dependent functions such as CTL-mediated lysis of target cells. Mac-1 (.alpha..sub.M.beta..sub.2) is distributed on neutrophils and monocytes, and its counter-receptor is also ICAM-1 (and possibly ICAM-2). Among other things, Mac-1 is the type 3 complement receptor (CR3) and binds the C3bi fragment. The third .beta..sub.2 integrin, P150,95 (.alpha..sub.X.beta..sub.2), is also found on neutrophils and monocytes, but seems of less importance. The a subunits of LFA-1, Mac-1 and P150,95 are also given the respective CD designations CD11a, CD11b and CD11c, while .beta..sub.2 is also denoted CD18, so that LFA-1 is CD11a/CD18 and Mac-1 is CD11b/CD18.
There are three known selectins, which were previously known as LECCAMs, and are now designated L-selectin (also called LECAM-1, Mel-14 or LAM-1), E-selectin (also called ELAM-1) and P-selectin (also called GMP140 or PADGEM). They have all been sequenced at the cDNA level and share sequence homology and motifs, including a lectin-like domain. L-selectin has a dual role: it is a homing receptor on T cells for the high endothelial venules of peripheral lymph nodes, and it is an adhesion molecule on neutrophils for endothelium (Hallmann et al., Biochem. Biophys. Res. Commun. 174:236 (1991), which is incorporated herein by reference in its entirety for all purposes). E-selectin and P-selectin are both induced on endothelium by cytokines, although with different kinetics. L-selectin is a counter-receptor on neutrophils for both E-selectin and P-selectin (Picker et al., Cell 66:921 (1991), which is incorporated herein by reference in its entirety for all purposes), although all three selectins probably have other counter-receptors as well. In particular, E-selectin binds the carbohydrate group sialyl Lewis x (sLex) (Lowe et al., Cell 63:475 (1990)), which is incorporated herein by reference in its entirety for all purposes), and while this carbohydrate is prominently presented on L-selectin (Picker et al., Cell 66:921 (1991)), it may occur on other proteins as well. E-selectin is expressed especially in cutaneous sites of inflammation and also serves as an adhesion molecule for skin-homing T cells that may contribute to the inflammation (Picker et al., Nature 349:796 (1991), which is incorporated herein by reference in its entirety for all purposes).
In various assays, antibodies to CD11a, CD11b, CD18, L-selectin and E-selectin all block binding of neutrophils to activated endothelial cells to a lessor or greater degree, but the most complete inhibition is generally achieved by the combination of an antibody to CD18 and an antibody to L- or E-selectin (see, e.g., Luscinskas, J. Immunol. 142:2257 (1989)), which is incorporated herein by reference in its entirety for all purposes). A recent but now widely accepted model accounts for these facts with a three step process of adhesion (Butcher, Cell 67:1033 (1991), which is incorporated herein by reference in its entirety for all purposes). In the first step, neutrophils reversibly bind to inflamed vascular endothelium via the selecting, which bind well under conditions of flow, causing the neutrophils literally to roll along the vascular wall. The neutrophils are then activated by a variety of stimulants surrounding or released by the endothelium, including IL-8, PAF and C5a. The activated neutrophils shed L-selectin and up-regulate Mac-1. In the final step, binding of Mac-1 to ICAM-1 and perhaps other counter-receptors on the endothelial cells allows stable adhesion and extravasation through the endothelium.
In principle, antibodies or other antagonists of the integrin and selectin adhesion molecules could abort this process, by preventing neutrophils from binding to endothelium and from extravasating into tissues. Hence such antibodies could be used to treat a great many different disease conditions of which inflammation is an important component.
For example, in animal models anti-CD18 antibodies, which bind to both LFA-1 and Mac-1, have been useful in reducing ischemia-reperfusion injury (see, e.g., Vedder et al., J. Clin. Invest. 81:939 (1988); Vedder et al., Proc. Natl. Acad. Sci. USA 87:2643 (1990); U.S. Pat. No. 4,797,277). They also reduce neutrophil-mediated damage in the lung in response to various insults (Doerschuk et al., J. Immunol. 144:2327 (1990) and Mulligan et al., J. Immunol. 148:1847 (1992)), including gram-negative sepsis (Walsh et al., Surgery 110:205 (1991)). In a rabbit model, anti-CD18 antibodies also protect from lethality due to meningitis (Tuomanen et al., J. Exp. Med. 170:959 (1990)). They may also be useful in preventing or treating organ transplant rejection because-they block T-cell function.
For example, injection of antibodies to L-selectin or E-selectin into rodents suppressed neutrophil accumulation within inflamed peritoneum (Jutila et al., J. Immunol. 143:3318 (1989) and Mulligan et al., J. Clin. Invest. 88:1396 (1991)). Intravital video microscopy revealed that an anti-L-selectin antibody strongly inhibits rolling of leukocytes along the vascular wall endothelium of mesenteric venules exteriorized from rabbits (von Adrian et al., Proc. Natl. Acad. Sci. USA 88:7538 (1991)). An anti-E-selectin antibody greatly reduced vascular injury induced by immune complex deposition in the skin or lungs of rats, and substantially reduced neutrophil accumulation at those sites (Mulligen et al., J. Clin. Invest. 88:1396 (1991)). Also, in a primate model of extrinsic asthma, an anti-E-selectin antibody greatly reduced neutrophil influx into the lung and associated late-phase airway obstruction after antigen inhalation (Gundel et al., J. Clin. Invest. 88:1407 (1991)).
Several antibodies including mouse DREG-55, mouse DREG-56 and mouse DREG-200 have been developed that bind to human L-selectin (Kishimoto et al., Proc. Natl. Acad. Sci. USA 87:2244 (1990), which is incorporated herein by reference in its entirety for all purposes). These antibodies partially or completely block the binding of human lymphocytes to peripheral lymph node high endothelial venules, and the binding of human neutrophils to stimulated human umbilical vein endothelial cells (Kishimoto et al., Blood 78:805 (1991), which is incorporated herein by reference in its entirety for all purposes). The capacity of these antibodies to block binding of neutrophils to endothelial cells indicates that the antigen to which they bind, L-selectin, may be an appropriate target for potential therapeutic agents.
Unfortunately, the use of non-human monoclonal antibodies such as mouse DREG-200 have certain drawbacks in human treatment, particularly in repeated therapeutic regimens as explained below. Mouse monoclonal antibodies, for example, have a relatively short circulating half-life, and lack other important immunoglobulin functional characteristics when used in humans.
Perhaps more importantly, non-human monoclonal antibodies contain substantial stretches of amino acid sequences that will be immunogenic when injected into a human patient. Numerous studies have shown that, after injection of a foreign antibody, the immune response elicited by a patient against an antibody can be quite strong, essentially eliminating the antibody's therapeutic utility after an initial treatment. Moreover, as increasing numbers of different mouse or other antigenic (to humans) monoclonal antibodies can be expected to be developed to treat various diseases, after the first or several treatments with any different non-human antibodies, subsequent treatments even for unrelated therapies can be ineffective or even dangerous in themselves, because of cross-reactivity. While the production of so-called "chimeric antibodies" (e.g., mouse variable regions joined to human constant regions) has proven somewhat successful, a significant immunogenicity problem remains.
To attempt to overcome immunogenicity problems several examples of humanized antibodies have been produced. The transition from a murine to a humanized antibody involves a compromise of competing considerations, the solution to which varies for different antibodies. To minimize immunogenicity, the immunoglobulin should retain as much of the human acceptor sequence as possible. However, to retain authentic binding properties, the immunoglobulin framework should contain sufficient substitutions of the human acceptor sequence to ensure a three-dimensional conformation of CDR regions as close as possible to that in the mouse donor immunoglobulin. As a result of these competing considerations, many humanized antibodies produced to-date show significant loss of binding affinity compared with corresponding murine antibodies. See, e.g., Jones et al., Nature 321:522-525 (1986); Shearman et al., J. Immunol. 147:4366-4373 (1991); Kettleborough Protein Engineering 4:773-783 (1991); Gorman et al., Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991); Tempest et al., Biotechnology 9:266-271 (1991); Riechmann et al., Nature 332:323 (1988) and EPO Publication No. 0239400) (each of which is hereby by reference in its entirety for all purposes).
Thus, there is a need for improved forms of humanized immunoglobulins specific for L-selectin antigen that are substantially non-immunogenic in humans, yet easily and economically produced in a manner suitable for therapeutic formulation and other uses. The present invention fulfills these and other needs.